Eukaryotic intracompartmental transport and secretory processes require fusion of vesicles with cellular membranes (1, 2, 3). A step leading to fusion of vesicles with cellular membranes is assembly of a tetrameric coiled-coil structure formed from sets of membrane proteins known as SNAREs (4) (soluble N-ethyl maleimide sensitive factor attachment protein receptors) (4). High-resolution structures are available for the final, post-fusion ternary SNARE complex that combines one vesicle protein (v-SNARE) with two target membrane proteins (t-SNARES). The SNAREs responsible for neuronal secretion are among the best-studied examples of this family of proteins. They include the v-SNARE VAMP (vesicle-associated membrane protein) that is located on the secretory vesicle membrane and two t-SNAREs—syntaxin (sx) and SNAP25 (synaptosomal-associated protein 25) present on the target membrane (5, 6).
SNAREs are responsible for selective transport between cellular compartments (7, 8). Alterations or damage to proteins or membranes that perform integral transport functions often produce disabling or irreversible consequences. Failure of the normal vesicular traffic (unless transient), which is the basis for intracellular transport and secretion, results in development of disease (9, 10). As a consequence, toxins that attack this machinery have significant effects on normal cellular processes.
Proteolytic cleavage of SNARE family components is currently known to be associated only with large microbial proteins from the genus Clostridium. These clostridial proteins require intracellular cleavage before they are released as smaller active proteolytic enzymes (23, 24, 25, 26). The present inventors have identified and isolated enzymes from scorpion venom that cleave also the SNARE complex proteins. These enzymes are unique from the clostridial enzymes with respect to their mechanism of cleavage, specific cleavage targets as well as their structure.